The long term objective of this proposal is to understand the function of the human retinoblastoma (Rb) gene(s). It is postulated that the loss of function in both alleles of this gene located in chromosomal region 13q14 is responsible for the development of both Rb and secondary malignancies in patients carrying the mutant Rb allele. The mechanism of tumor development may also be similar in patients with osteosarcomas without any hereditary susceptibility to retinoblastoma. In addition the same mechanism of tumor formation has been implied for the development of Wilms' tumor. Specific aims include determining changes in human Rb and second primary tumors following the addition of a 13 chromosome containing the normal wild type Rb allele. Parameters tested in such hybrid cells will include changes in ability to produce tumors in nude mice, differences in the capacity to form colonies in semisolid medium, changes in specific protein markers and alterations in the expression of oncogenes which are known to be amplified and/or highly expressed in the original parental cells. In addition fibroblasts from patients with the hereditary deletion and non-deletion forms of Rb will be transformed with various chemical and physical agents to determine if the loss of the normal wild type Rb allele at 13q14 results in the transformation of these cells. Such studies will provide further evidence that the retinoblastoma gene functions normally to suppress tumorigenicity and that loss of function within both alleles is responsible for tumor formation. Expression of oncogenes in these transformed cells as well as the specific role of the N-myc gene in the development of retinoblastoma and secondary malignancies will be examined. Methodologies will include microcell fusion, 2-dimensional gel electrophoresis, restriction fragment length polymorphism analysis, esterase D assays, karyotypic analysis and standard recombinant DNA technology. These studies should lead to a much better understanding of a class of human cancer susceptibility genes which are recessive and appear to normally function to "suppress" tumor development. Information gained from these investigations should not only further our understanding of human cancer development but also could suggest clues to possible ways of preventing malignant progression.